research article pollen viability and pollen germination...

Research ArticleIn Vitro Pollen Viability and Pollen Germination in CherryLaurel (Prunus laurocerasus L.)

Melekber Sulusoglu1,2 and Aysun Cavusoglu1,2

1 Arslanbey Agricultural Vocational School, Kocaeli University, 41285 Kocaeli, Turkey2Department of Horticulture, Graduate School of Natural and Applied Sciences, Kocaeli University, 41380 Kocaeli, Turkey

Correspondence should be addressed to Melekber Sulusoglu; melekber [email protected]

Received 2 July 2014; Revised 2 September 2014; Accepted 18 September 2014; Published 22 October 2014

Academic Editor: Nnadozie Oraguzie

Copyright © 2014 M. Sulusoglu and A. Cavusoglu. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability andpollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.). Two pollen viability tests, TTC (2,3,5-triphenyltetrazolium chloride) and IKI (iodine potassium iodide), were used. Pollen traits of genotypeswere studied using an in vitromediumcontaining 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step,the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rateswere different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation(𝑟2 = 0.0614 and 𝑟2 = 0.0015, resp.). Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viabilityestimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germinationrates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose andthe results showed that pollen germination rates increased after 6 hours of being placed in culture media.

1. Introduction

The development of reliable methods for determining thefunctional quality of pollen helps in monitoring pollenvigor during storage, genetics and pollen-stigma interactionstudies, crop improvement and breeding, and incompatibilityand fertility studies [1–4]. The quality of pollen is assessedon the basis of viability and vigor of the pollen grain. Pollenvigor refers to the speed of germination of pollen grains andthe rate of pollen tube growth [5]. In vitro pollen germinationtests have been used to determine the germination percentageof pollen and can also be used for assessing pollen vigor bymonitoring the rate of germination over a period of timeor the length of pollen tubes [6]. Pollen which could notgerminate usually shows poor tube growth which is likelyto be ineffective in causing fertilization. In the breeding offruit species it is very important to use a suitable methodfor determining pollen viability [7]. In vitro germination testshave been used to indicate viability of pollen [8]. In general,there is a linear relationship between pollen viability and

germination capability in many fruit species [1]. However insome cases, different results may be obtained in cultivars withstaining tests. Therefore, to determine the actual amount ofviable pollen, germination tests are necessary.

The material in this study is cherry laurel (Prunus lau-rocerasus L.), belonging to Rosaceae family. It is one of thefruit species native to southeastern Europe and Asia Minoraround the Black Sea in the Caucasus and Transcaucasiamountains of Anatolia, Bulgaria, and Serbia [9]. Prunuslaurocerasus is an evergreen plant with small cherry fruits, afew centimeters in diameter. The fruit color is red to blackwhen ripe. Before maturity fruits are astringent but becomesweet and reasonably pleasant when fully ripe. The stoneswhen ground into powder are very good for bronchitis [10].Cherry laurel fruits are considered a significant source ofphenolic compounds and anthocyanins [11, 12], and it is oneof the most widely used ornamental plants for landscaping[13].

It is widely spread in the northern part of Turkey andthere are many cultivars which show different traits. As an

Hindawi Publishing Corporatione Scientific World JournalVolume 2014, Article ID 657123, 7 pageshttp://dx.doi.org/10.1155/2014/657123

2 The Scientific World Journal

alternative fruit, with a different taste, it has commercialvalue in Turkey and many gardens are planted in selected 4cherry laurel genotypes. Some breeding and cultivation forornamental studies have just started in United States andEurope [14–16]. Cherry laurel yield amount varies from yearto year; that is, the trees tend to have high production inone year followed by low production the following year. Themost important factors affecting fruit set are late spring frost,rainy spring weather during bloom, heat stress, and drought[10]. Breeding to obtain cultivars with high fruit quality hasrarely been attempted [12, 17–19]. Cherry laurel is a self-incompatible species and needs cross-pollination for fruit setaccording to our observations. There is usually severe fruitdrop leaving no fruit on a shoot. One of the important factorsfor fertilization success is pollen viability and germination;therefore pollen performance may have a significant role inpollination for adequate fruit yield [20, 21]. Information onpollen biology of cherry laurel is very limited and for thispurpose, the aim of the study was to determine the pollenviability and in vitro germination rate of cherry laurel pollen.

2. Materials and Method

The study was conducted over 2011-2012 to evaluate thepollen quality of seven cherry laurel genotypes. These sevengenotypes were selected from the selection study where 40cherry laurel genotypes were evaluated between 2008 and2010 [22]. Unopened flowers were collected at balloon stagein April from all sides of one tree (each genotype/one tree).Petals and sepals were separated and anthers were isolatedfrom flower buds and placed on a black paper under anincandescent lamp on a table overnight. In the next day, thepollen grains were collected in a small glass vial.

Pollen viability was estimated using two staining tech-niques, IKI (iodine potassium iodide) [23] and TTC (2,3,5-triphenyl tetrazolium chloride) [24–26]. 1% TTC (0.2 g. TTCand 12 g sucrose dissolved in 20mL distilled water) was usedin the first step and a drop of the mixture was dropped ona microscope slide and the pollen spread with a slim brushand covered with a coverslip. Pollen viability counts weremade after two hours; then pollen was placed on a TTCsolution. Pollen grains stained that orange or bright red colorwere counted as viable. In the second staining method, 1 gpotassium iodide and 0.5 g iodine were dissolved in 100mLdistilled water for the IKI solution. Pollen viability countswere made five minutes after pollen was placed on an IKIsolution. Pollen grains stained dark (dark red or brown color)were counted as alive.

Stain tests are faster and easier than pollen germinationtests but in some cases germination tests are necessary toobserve the actual viability of pollen. Studies on pollenviability in Prunus that in vitro germination by an agarose-sucrose medium gave good results [27] and we used theagarose-sucrose medium method for the pollen germinationtreatments. In the first step of pollen germination studies,different sucrose concentrations (0, 5, 10, 15, and 20%) wereadded into media together with 0.5% agar and 5 ppm boricacid (H

3BO3) to determine the effects of sucrose concentra-

tions. Pollen germination was observed at the end of 24-hour

incubation period. In the next study pollen germination ratewas determined during the 48-hour incubation period, in thebest level of sucrose (15%), in an oven, at 22∘C in the dark.Thegerminated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hoursafter incubation.

In both germination treatments, the pollen grains wereconsidered germinated when the pollen tube length reachedpollen diameter [28].The experiments were designed as com-pletely randomized blocks with three replications. Randomlyselected visual areas, including about 100 pollen grains, werecounted in each replicate. All observations of viability andgermination were made at ×100 magnification using a lightmicroscope.

In both years (2011 and 2012), pollen viability andgermination rates were very close to each other. All thedata collected were combined and the means were usedin the statistical analysis. Statistical analysis was performedin the Minitab 17 Statistical program. One-way ANOVAwas used for the analysis of viability and germination ratesand different means were compared with Duncan’s MultipleRange Test at 𝑃 < 0.05. The values for viability and pollengermination were transformed to arcsin square root valuesbefore statistical analysis.

Correlation analysis was performed to determine therelationship between pollen viability and pollen germination(after 48 hours). These correlations were evaluated usingMINITAB 17 statistical program (𝑃 ≤ 0.05) for TTC and IKIstaining tests.

3. Results and Discussion

The results of TTC and IKI staining tests are shown in Table 1.The interaction of genotypes × staining tests was statisticallysignificant and there were slight differences in the pollenviability percentages of genotypes in both staining tests. IKIgave the highest pollen viability rates and the genotypesstained better with IKI solution than pollen stained with 1%TTC (Figures 1(a) and 1(b)).

The cherry laurel genotypes exhibited differences inpollen viability depending on the staining method. In theTTC test, genotype 36 had the highest pollen viability(97.69%) but this result was not different statistically fromother genotypes. According to viability rates obtained withIKI, the lowest percentage of viable pollen was found to be81.25% in genotype 4 and this result is statistically significant(Table 1). Similar differences according to genotypes andstaining tests have been observed in other studies [27, 29].Determining the viability of pollen grains is an easy methodto increase the efficiency of breeding programs and forselection of suitable pollinizers while orchards are beingestablished [30, 31]. In this study, pollen viability of thegenotypes was very high in both staining tests; therefore, allgenotypes can be considered as good pollinators [32].

Generally pollen grains require water for the formationof a pollen tube and excellent germination was obtainedin sugar media with or without a lower percentage ofagar [33]. Depending on the sucrose level, cherry laurelpollen exhibited different germination rates. The interactionbetween sucrose concentration and genotypes of cherry laurel

The Scientific World Journal 3

Table 1: Pollen viability of cherry laurel genotypes determined by TTC and IKI tests.

Genotypes∗ TTC (%) IKI (%) Mean of genotypes (%)GT4 95.29abA 81.25bB 88.27GT5 86.87abA 93.24aA 90.06GT7 88.78abB 97.70aA 93.24GT24 88.89abB 97.73aA 93.31GT31 95.59abA 97.30aA 96.45GT34 93.95abA 92.98aA 93.47GT36 97.69aA 98.77aA 98.23Mean 92.44 94.14 General mean: 93.29∗Genotypes names were given as in the selection study [22].Values within the same column (genotypes) with different lower-case letters and values within the same row (staining tests) with different capital letters aresignificantly different (𝑃 < 0.05).

(a) (b)

Figure 1: TTC (a) and IKI (b) painted pollen of cherry laurel (×400).

was not statistically significant, while the effect of sucroseconcentration was found statistically significant at 𝑃 < 0.05level. Pollen germination rates increased up to 15% sucroseconcentration and decreased at 20% sucrose concentrationexcept in genotypes 4 and 34 (Table 2).

Pollen grains did not develop a pollen tube in 0% and5% sucrose including media, or there was only a verysmall tube and tube elongation increased with increasingamount of sucrose in the culture media (Figures 2(a), 2(b),2(c), and 2(d)). Pollen tube formation was inhibited againin 20 g/L sucrose including media, and pollen tube lengthwas decreased too (Figure 2(e)). In Figure 3, pollen tubeformation of cherry laurel was given (Figure 3(a)) and unger-minated pollen without a pollen tube (Figure 3(b)) in theculture media was shown.

With genotype 34, 10% sucrose concentration gave thehighest germination percentage and increasing concentra-tion of sucrose affected negatively the germination rate ofthe pollen. Pollen germination rates were increased withthe increasing concentrations of sucrose in genotype 4.Differences among sucrose means were found statisticallysignificant and the lowest germination percentages wereobserved in sucrose free media in all genotypes. Previousstudies have shown that the most suitable concentration ofsucrose for stone fruit pollen germination under in vitro

conditions was 15% or 20% sucrose [27, 34–37]. These resultsare in agreement with our findings.

According to personal observations, pollen grain struc-ture exhibited high morphological homogeneity and it couldbe used as a good property for fertilization biology [38].

The correlations between pollen viability and germina-tion tests were also determined and were not significant inTTC and IKI tests (𝑟2 = 0.0614 and 𝑟2 = 0.0015, resp.)(Figure 4). Previous studies to determine the relationshipbetween pollen viability and germination tests in fruit speciesshowed different results. For example, viability as determinedby TTC staining tests in apple, pear, peach, and grape pollendid not show good correlation with the actual germinationrate [25]. However, Werner and Chang [34] observed thatthe correlation between the MTT (Baker’s and X-Gall) staintest and pollen germination in peach was significant but theIKI test was not. Parfitt and Ganeshan [27] have establishedthat the pollen stain tests (including TTC) are not reliableor consistent and are not positively correlated with in vitrogermination tests. These results are in agreement here withour findings.These stainsmay therefore be used to determinepollen viability in these species to provide only a roughestimate of viability.

Incubation time of pollen is important for in vitro pollengermination and in the second step of the germination


(a) (b)

(c) (d)

(e)

Figure 2: Pollen germination in media containing different sucrose concentrations after 24 hours of germination ((a) 0%, (b) 5%; (c) 10%;(d) 15%, and (e) 20% sucrose) (×100).

Table 2: Effect of sucrose concentrations on agar plate media on pollen germination of cherry laurel.

Genotypes Sucrose (%) Mean of genotypes0 5 10 15 20

GT4 26.84 38.33 56.31 64.98 69.36 51.16GT5 18.68 37.71 41.78 69.66 60.47 45.66GT7 15.86 32.84 50.80 67.84 59.92 45.45GT24 44.30 52.97 56.40 71.03 53.81 55.70GT31 23.32 33.84 47.37 69.51 47.32 44.27GT34 32.56 34.98 65.46 60.75 55.71 49.89GT36 39.90 47.04 55.75 72.13 68.26 56.62Mean 28.78c 39.67bc 53.41ab 67.99a 59.26a General mean: 49.82Values with different lower-case letters are significantly different at 𝑃 < 0.05.


(a) (b)

Figure 3: Pollen tube growth on germinated pollen (a); ungerminated pollen without a pollen tube (b) (×400).

70

80

90

100

110

40 55 70 85 100 115

Polle

n vi

abili

ty in

TTC

(%)

Pollen germination (%)

70

80

90

100

110

40 55 70 85 100 115

Polle

n vi

abili

ty in

IKI (

%)

Pollen germination (%)

y = 0.1048x + 85.134 y = −0.0253x + 95.901

R2= 0.0015R

2= 0.0614

Figure 4: Correlation between the pollen staining test and pollen germination rates.

studies, incubation time effects were significant (Table 3).Pollen tubes began to grow approximately 30 minutes afterbeing placed in the growing medium and reached maximumgermination at the end of 48 hours (Table 3). As reportedin another study, the highest germination was obtained after48 hours of incubation period for cherry cultivars [29]. Dif-ferences between 24-hour and 48-hour pollen germinationrates were statistically not significant but pollen tube lengthsdramatically increased over time (data not presented). 24hours may therefore be enough for germination of cherrylaurel pollen.

In the 48-hour germination period, pollen germinationrates were different among the genotypes and this wasstatistically significant at 𝑃 < 0.05 (Table 3). Genotype36 germinated faster than other genotypes and gave thehighest germination rate (79.14%) at the end of 48 hours.According tomean germination values, this genotype showedstatistically higher germination rates than other genotypesover the counting period (Figure 5). There are reports in theliterature on the occurrence of variation in in vitro pollen

germination of fruit cultivars, such as sweet cherry, caprifig,and medlar [29, 39, 40], and the results presented herecorroborate those studies.

4. Conclusions

In this study, results showed pollen viability and germinationrates in various conditions for genotypes of cherry laurel.Results of this study indicated that TTC and IKI stainingtests could be used to determine the viability of cherry laurelpollen while TTC gave results close to real germination rates.The results of viability tests were close among the cherrylaurel genotypes and viability rates were very high. 15%sucrose was the best sucrose concentration for germinationand the appropriate germination time was 24 hours by bothobservation and counting. The results presented here are thefirst observation on the cherry laurel pollen viability andgermination rate that will help in cherry laurel reproductionand artificial pollination studies.


Table 3: Germination rate of cherry laurel genotypes within 48 hours.

Genotypes Germination time (hours) Mean of genotypes1 4 6 10 12 24 48

GT4 20.92 33.11 37.71 58.49 60.45 65.93 76.63 50.46abGT5 21.66 29.83 41.27 46.06 49.28 52.15 61.11 43.05bcGT7 19.01 33.00 40.12 41.94 53.06 56.78 65.37 44.18bcGT24 34.61 49.77 56.16 56.53 56.53 71.04 75.31 57.14abGT31 26.78 41.19 43.78 46.21 60.37 67.55 68.41 50.61abGT34 16.11 16.40 24.89 25.8 27.26 43.38 61.82 30.81cGT36 25.50 60.91 62.13 63.56 69.91 79.01 79.14 62.88aMean 23.51d 37.74cd 43.72c 48.37bc 53.84bc 62.26ab 69.68a 48.45Values with different lower-case letters are significantly different at 𝑃 < 0.05.

0102030405060708090

100

1 4 6 10 12 24 48

(%)

Incubation time (hours)

GT36GT34GT4GT7

GT31GT5GT24

Figure 5: Germination percentage of cherry laurel genotypes overthe incubation period.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

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